Intercellular communication mediated by extracellular vesicles (EVs) has recently attracted significant attention, and may contribute to diverse physiological processes including immune regulation and cancer metastasis. EVs are comprised of two major classes based upon their mechanism of biogenesis: microvesicles are shed directly from the plasma membrane, while exosomes are formed by intra-lumenal budding during multi-vesicular endosome (MVE) biogenesis (Colombo et al., 2014; van Niel et al., 2018) (Figure 1A). EVs contain a wide range of molecules, including antigens, signaling proteins, lipids, and microRNAs (miRNAs) that appear to induce significant biological changes in recipient cells. EV miRNAs released by both tumor and mesenchymal cells are of particular interest, as they may play important roles in metastasis (Becker et al., 2016). EV miRNAs secreted by hypothalamic stem cells may regulate aging in mice (Zhang et al., 2017). Moreover, EVs have attracted a great deal of attention due to their potential use in diagnostics and therapeutics (Becker et al., 2016). Despite their importance, how miRNAs are specifically packaged and released via the EV pathway is poorly understood. The biological significance of EVs also remains controversial, in part due to a lack of understanding of the molecular mechanisms that regulate their formation and release. Here we describe the development of a novel CRISPR screening platform using artificially barcoded miRNAs (bEXOmiRs) that we applied systematically to identify genes involved in EV biology. We show that EV release is regulated tightly by Glycogen Synthase Kinase three and Wnt signaling, and report new players in MVE exocytosis.

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Cell culture, antibodies and immunofluorescence--HEK293T cells were grown in Dulbecco's modified Eagle's media (DMEM) containing 10% fetal bovine serum, 2 mM L-glutamine, and penicillin (100 U/ml)/streptomycin (100 μg/ml). Wild type (WT) and Cas9-expressing (Morgens et al., 2017) K562 cells were grown in RPMI 1640 Medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 1 mM sodium pyruvate and penicillin (100 U/ml)/streptomycin (100 μg/ml). All cell lines were cultured at 37°C with 5% CO₂; cells were routinely tested for Mycoplasma using either a MycoAlert Mycoplasma Detection kit (Lonza LT07-318) or PCR. Their identity was assumed to be determined by ATCC. Extracellular vesicle-free media was prepared by overnight ultracentrifugation of RPMI supplemented with 20% FBS at 100,000 X g in a 45Ti rotor, as described (Théry et al., 2006).

Transfections of HEK293T cells plated in 10 cm dishes were carried out using 1.5 μg of bEXOmiR-encoding plasmid DNA (pEGFP-bEXOmiR) mixed with 1 ml serum-free DMEM and 30 μL polyethylenimine (PEI, 1 mg/ml, pH 7.0, PolySciences Inc.) and incubated at room temperature for 30 min. The mixture was added to cells that were grown for 48 hr before EV isolation. For Bafilomycin-A1 (Sigma-Aldrich) treatment, the drug was added to cell media at 10 nM and cells were cultured for 20 hr before EV collection.

Mouse monoclonal anti-α-Tubulin was from Sigma-Aldrich, rabbit polyclonal anti-ARHGEF18 was from GeneTex and anti-Rab27A was from Synaptic Systems. Mouse monoclonal anti-CD81, anti-β-Catenin and anti-Golgin 97 were from BD biosciences. Polyclonal anti-GDIβ was previously described (Dirac-Svejstrup et al., 1994). Rabbit polyclonal anti-Flotillin-1 antibody was from Novus Biologicals (NBP1-79022). Polyclonal anti-Calnexin and monoclonal anti-HSC70 antibodies were from Santa Cruz. Mouse monoclonal anti-LAMP1 antibody culture supernatant was from Developmental Studies Hybridoma Bank (University of Iowa, Iowa city, IA). Mouse monoclonal anti-LBPA and anti-CD63 antibodies were from EMD Millipore (MABT837) and DHSB (H5C6), respectively.

Immunofluorescence-- HeLa cells were transfected with fluorescently labeled siRNAs (DY547 label; Dharmacon) using DharmaFECT one transfection reagent, cultured for 48 hr, and replated on glass coverslips. To visualize K562 cells, cells were attached to glass coverslips using cytospin (Shandon) at 800 rpm for 5 min. Cells were then fixed with 3.7% (v/v) paraformaldehyde for 15 min, washed and permeabilized/blocked with 0.1% saponin/1% BSA-PBS before staining with anti-LAMP1 antibody followed by goat anti-rabbit Alexa 488 (1:2,000). Nuclei were stained using 0.1 µg/ml DAPI (Sigma-Aldrich). Coverslips were mounted on glass slides with Mowiol and imaged using a spinning disk confocal microscope (Yokogawa) with an electron multiplying charge-coupled device (EMCCD) camera (Andor, UK) and a 100 × 1.4 NA oil immersion objective. Finally, unbiased quantification of CD63-positive vesicles was performed using CellProfiler software (Carpenter et al., 2006).

Immunoblotting--HEK293T or K562 cells were harvested after 24–48 hr and lysed in lysis buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM MgCl2, and 1% Triton X-100) supplemented with protease inhibitors. After centrifugation at 12,000 g for 10 min, protein concentrations were measured in the cleared lysates. Equal amounts were resolved by SDS-PAGE followed by immunoblot analysis. EV final pellets were resuspended in SDS-PAGE loading dye (50 mM Tris-HCl pH 6.8, 6% glycerol, 2% SDS, 0.2% bromophenol blue), heated briefly at 95°C and resolved by 10% SDS-PAGE. After transfer to nitrocellulose and antibody incubation, blots were detected with ECL western blotting detection substrate or visualized using LI-COR Odyssey Imaging System. Isolation of cytosolic fractions from cell post-nuclear supernatants was performed as described (Lu and Pfeffer, 2013). For optimal detection of EV fraction markers (such as CD81), no reducing agent was added to the SDS-PAGE loading dye. Immunoblot signals visualized using LI-COR were analysed using ImageJ and presented as average ±SEM.

EV isolation--K562 suspension cell cultures were seeded at 5 × 10⁵ cells/ml in EV-free RPMI and grown for 24–48 hr. Cell cultures were centrifuged at 300 × g, 5 min to pellet cells, and supernatants were centrifuged again at 2100 × g, 20 min to pellet dead cells. The resulting supernatant was spun again at 10,000 × g, 30 min to pellet cell debris and large apoptotic bodies; for large scale screen cultures this step was replaced by filtration through 0.45 µm filter units (Thermo Scientific). After this, filtration through 0.22 µm filter units (Thermo Scientific) was performed. Filtered media were ultracentrifuged for 70 min at 100,000 × g in 45Ti rotor to pellet extracellular vesicles. The supernatant was discarded and the pellet re-suspended in phosphate-buffered saline (PBS) and centrifuged again for 70 min at 100,000 × g in TLA100.2 rotor. Discontinuous sucrose gradient flotation was as described (Shurtleff et al., 2016). EV pellets were frozen in liquid nitrogen and stored at −80 ˚C prior to further processing.

Nanoparticle tracking analysis--EVs were purified from control and CRISPR KO K562 polyclonal cell cultures as described (see EV isolation). After final ultracentrifugation, pelleted EVs were resuspended in PBS and analyzed on a NanoSight NS300 (Malvern Panalytical) using a sCMOS camera. Three, 60 s 25 Hz movies were taken per sample. Data were analyzed using NTA 3.1 software (Malvern Panalytical).

Flow cytometry analysis of LAMP1--K562 cells were treated with either DMSO or CHIR99021. After 24 h cells were harvested and washed before fixation in 2% paraformaldehyde, permeabilization and staining with anti-LAMP1 and Alexa Fluor 488 goat anti–mouse antibodies (Invitrogen). Quantification of LAMP1 total fluorescence was performed using a BD Accuri C6 flow cytometer and data were analyzed using Flowjo software (Tree Star).

Total internal reflection fluorescence (TIRF) microscopy--HeLa cells stably expressing pHluorin-CD63 were transfected with fluorescently labeled siRNAs (DY547 label; Dharmacon) using DharmaFECT one transfection reagent, cultured for 48 hr, and replated on chambered coverglass (Thermo Scientific). After 24 hr, cells containing fluorescent siRNAs were imaged on an inverted microscope (Nikon Eclipse Ti) equipped with an electron-multiplying charge-coupled device camera (ANDOR iXon^EM+), an Apo TIRF 100X/1.49 oil DIC objective, and Nikon Perfect Focus System. Time lapse videos were acquired at 3 Hz with Nikon NIS Elements software. All imaging experiments were performed at 37°C in a humidified incubator (5% CO₂). Fusion events for each cell were detected and quantified as sudden increases in fluorescence above the plasma membrane background as described (Verweij et al., 2018). At least 20 cells were imaged per condition in two independent experiments.

bEXOmiR design--Initial development of bEXOmiRs was based on previous artificial shRNAs designs (Chang et al., 2013) incorporating endogenous sequences derived from the putative exosomal microRNA hsa-miR-601. Specifically, a seven nt motif (GGAGGAG, ‘EXO motif’) together with a contiguous loop sequence (both derived from hsa-miR-601) were placed adjacent to a 15 nt random sequence (barcode) with perfect base complementarity, positioned at the base of the miRNA stem region (Figure 1—figure supplement 1A). The resulting precursor hairpin was flanked by hsa-miR-30a context sequences. For the initial bEXOmiR test library, a custom C-script was written to design barcode sequences based upon specific rules: 40–60% GC content, minimum Hamming distance of 4 or more between any two barcodes, and absence of homopolymers (AAAA, UUUU, CCCC, etc.) and restriction enzyme cut sites used for cloning purposes. With this strategy, 2500 bEXOmiRs were designed, and two versions of each were used, one with an additional A-C mismatch added to ensure proper primary-miR processing (Chang et al., 2013) for a total of 5000 bEXOmiRs.

To generate ~25,000 bEXOmiRs used in the MMM screen sublibrary, 15 base-pair barcodes were similarly generated with a minimum Hamming distance of four, moderate GC content (40–60%), and absence of homopolyers (GGGG, AAAA etc) and restriction sites. The first base-pair was altered to create a mismatch at the base of the hairpin to ensure proper processing (either A-C, T-C, G-A, or C-A). For subsequent sublibraries, this ~25,000 bEXOmiR set was filtered for either bEXOmiRs that failed to be detected in WT exosome samples (counts less than 10) or were detected at such high representation in WT exosomes as to interfere with sequencing depth (count >10,000). Additional barcodes were randomly generated as above for larger libraries.

Stem-loop RT-PCR and qRT-PCR--For bEXOmiR and endogenous miRNA detection in cellular or EV fractions, RNA was extracted from intact HEK293T or K562 cells, or isolated EVs using TRIzol, according to the manufacturer. EV RNAs (200–500 ng) were reverse transcribed into cDNA using Stem-Loop primers and SuperScript III FirstStrand Synthesis System (Life Technologies) according to a pulsed RT protocol (Varkonyi-Gasic and Hellens, 2011). Stem-loop RT primers are summarized in Supplementary file 3. qRT-PCR experiments used a qPCR mix recipe: 1X Phusion HF buffer (NEB), 200 µM dNTPs, 1X SYBR green I (Invitrogen), HighFidelity DNA Polymerase (M0530S, NEB), and 100 nM each primer. The following programs were used: (98°C, 3’, then 40X cycles of 98°C 15 s, 60°C 20 s, 72°C 3 s). A synthetic spike-in RT RNA (Supplementary file 3) was added to each EV sample to control for variability in the starting material and the downstream RNA isolation step. The sequences of PCR primers are summarized in Supplementary file 3. qPCR was run in a BioRad CFX96 Real-Time System. PCR amplification products were analyzed on 4% agarose gels stained with ethidium bromide. For qPCR experiments related to Figure 7, total cell RNA was reverse transcribed into cDNA using oligo(dT)20 primers and SuperScript III FirstStrand Synthesis System (Life Technologies). qPCR was performed using iTaq Universal SYBR Green Supermix (BioRad) using gene-specific primers (Supplementary file 3) and run in a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). Transcript levels relative to HPRT1 were calculated using the ∆Ct method.

RNase protection assay--K562 cells expressing bEXOmiRs were grown for 24 hr in RPMI and EVs were isolated from conditioned media. EVs were then treated with RNase A (10 µg/ml in PBS)±Triton X-100 (0.25%), or with PBS alone, for 30 min at room temperature. RNase inhibitor (150 U/ml, Thermo Fisher Scientific) was then added and EV-RNA isolated with TRIzol reagent (Life Technologies).

Construction of sgRNA-bEXOmiR plasmid libraries--Oligonucleotides encoding sgRNAs (Morgens et al., 2017) coupled to individual bEXOmiRs were synthesized as pooled libraries (Agilent). BlpI and EcoRI restriction sites were introduced between sgRNA and bEXOmiR sequences. Focused sublibraries were PCR-amplified with primer sequences common to each sublibrary, digested with BstXI and MfeI enzymes, ligated into BstXI/EcoRI-digested pMCB306 vector, and transformed into electro-competent cells (Lucigen). Plasmid DNA was isolated using a QIAGEN kit. Pooled plasmids were then digested with BlpI and EcoRI, and an expression cassette encoding a puromycin-resistance gene and a GFP-ORF separated by a T2A sequence under EF-1 alpha promoter, cut with the same restriction enzyme pair, was inserted between sgRNA and bEXOmiR sequences. This generated nine focused sublibraries (48, Supplementary file 4), which together contained ~210,000 elements targeting ~20,000 genes with ~13,000 non-targeting and safe-targeting controls (Morgens et al., 2017); 10 sgRNA per gene, each sgRNA associated to a unique bEXOmiR).

Lentivirus production and cell infection--Lentivirus production and infection were performed as described (Kampmann et al., 2014). Briefly, HEK293T cells were co-transfected with bEXOmiR libraries or individual sgRNA vectors and third generation packaging plasmids (pVSV-G, pMDL, pRSV) using polyethylenimine. Lentivirus was harvested after 48 h-72 h and filtered through a 0.45 μm filter (Millipore). A Spinfection method was used to deliver sgRNA-bEXOmiR lentiviral plasmid sublibraries into K562 cells. Briefly, K562 cells were re-suspended in lentiviral supernatant supplemented with 8 µg/ml polybrene, distributed into 6-well plates, and centrifuged for 2 hr at 1000 × g, 33°C. Lentiviral supernatant was removed and cells were grown in fresh RPMI. After 48 hr recovery, 1.75 µg/ml of puromycin was added to select the cells for stable integration of the virus. Selection was monitored by flow cytometry and puromycin was removed from the media once >95% of the cells were GFP-positive.

CRISPR/Cas9-bEXOmiR screens in K562 cells--For genome-wide coverage, a total of nine, focused CRISPR/Cas9 deletion screens using nine different sublibraries of sgRNA-bEXOmiR associations (Supplementary file 4) were performed. WT and Cas9-expressing K562 cells were infected at low MOI (<1). Transduced cells were selected and expanded in puromycin-supplemented media over 5–7 days before conducting experiments at 8–10 days post-infection. Initially, we performed two independent pilot screens focused on membrane trafficking, mitochondrial and motility genes (MMM screen). Subsequent screens were conducted in duplicate to further minimize technical variation observed in previous screens. Screens were started at 10,000–15,000 fold sublibrary coverage (elements per ml of cell culture) to ensure maximal bEXOmiR representation in EVs but also to minimize variability due to downstream loss of barcodes during RNA isolation and fractionation. Isolation of EVs was performed 48 hr later using protocol P2 (Figure 1—figure supplement 1B). Cellular fractions with at least 1000-fold sublibrary coverage were collected at the start (0 hr) and end (48 hr) of each screen.

bEXOmiR isolation and purification from screen EV RNA--Total RNA was extracted from each EV sample using TRIzol reagent (Life Technologies), as described (Lässer, 2013). The resulting RNA pellet was re-suspended in nuclease-free water and equal amount of 2x RNA loading dye was added (90% formamide, 50 mM HEPES, bromophenol blue, xylene cyanide). Fractionation of total RNA was carried out on denaturating polyacrylamide gels containing 7% urea/15% acrylamide in Tris-borate-EDTA buffer (TBE). Custom 21 and 24 bp single-stranded RNA markers were used to indicate approximate miRNA migration on a gel from which gel band excision was monitored by UV shadowing. miRNAs were then gel-purified, reconstituted in nuclease-free water, and stored in −80 ˚C.

Small RNA library preparation and deep sequencing--bEXOmiR sequencing libraries from isolated EV miRNA was prepared using a modified protocol from TruSeq Small RNA Library Preparation Kit (Illumina). For each library,~1 µg of fractionated small RNA was ligated first to a 3’-adapter and then to 5’-adapter, followed by reverse transcription with custom primer (Supplementary file 3) containing a fragment complementary to the EXO motif in bEXOmiRs (to specifically enrich for bEXOmiRs in the final sequencing library). Resulting cDNA was then uniquely indexed by PCR to generate the final sequencing library. Initial sequencing trials showed some rRNA contaminant fragments (specially 45S5) that were predominant in our sequencing data, which compromised sequencing depth. To remove rRNA 45S5, a specific hairpin oligo blocker (Supplementary file 3) containing a sequence complementary to the predominant rRNA fragment was incubated after ligation of the 3’ adapter to the miRNA (Roberts et al., 2015). This oligo blocked subsequent ligation of the 5’ adapter to the contaminant rRNA fragment, thus excluding it from later steps. After PCR amplification, indexed samples were separated on a 4.5% low melting point agarose gel and products corresponding to 140 bp were purified using a MinElute PCR Purification Kit (Qiagen). RNA concentration was determined by Qubit fluorometer (Thermo Fisher Scientific). Pooled libraries were denatured with NaOH and diluted to 2.3 pM according to the Illumina protocol, and sequenced using a NextSeq 500/550 High Output v2 kit on NextSeq 500 system (Illumina).

Sequencing data analysis--Sequencing of sgRNAs from DNA was trimmed and aligned using Bowtie version 1.1.2 with zero mismatches tolerated (Morgens et al., 2016). All counts from multi-mapping reads were used. For sequencing of barcodes from RNA, reads were trimmed to 14 base-pairs and aligned to the libraries using Bowtie version 1.1.2 with two mismatches tolerated and all multi-mapping reads used. Supplementary file 5 and 6 contain bEXOmiR and sgRNA sequencing counts derived from each duplicated screen. Gene-level effects and an associated log-likelihood ratio (confidence score) were then calculated with casTLE as previously described (Morgens et al., 2016). For each gene, casTLE combines measurements from 10 different bEXOmiRs (associated with the sgRNAs targeting each gene) and gives an effect size estimate for the gene perturbation (gene K.O.) as well as a p-value associated with that effect. casTLE estimates the maximum possible phenotype such that targeting elements are most likely to be found between this phenotype and zero. Additionally, casTLE allows data from multiple screen types or from replicates of the same screen type to be compared and combined by finding a single effect size consistent with all data (Morgens et al., 2016). Briefly, low count (<10) bEXOmiRs were filtered, and a median-normalized log count ratio was calculated for each barcode. Distributions of barcode enrichments corresponding to each gene were compared to the distribution of nontargeting and safe-targeting sgRNAs. P-values were then calculated by permuting bEXOmiRs corresponding to targeting sgRNAs. Code is available at https://bitbucket.org/dmorgens/castle (Copy archived at https://github.com/elifesciences-publications/dmorgens-castle).

Nanostring profiling was performed with the nCounter Human v3 microRNA expression assay. Control, SMPD3 and ARHGEF18 CRISPR-KO K562 polyclonal cell lines were seeded at 2.5 × 10⁵ cells/ml in EV-free RPMI medium and grown for 48 hr before EV isolation. RNA from cellular and exosomal fractions was extracted using Trizol as described (Lässer, 2013). EV RNA was resuspended in 4 µL H₂O and 3 µL (~300 ng) from each sample were used in the assay; 200 ng cellular RNA was used. RNA integrity was confirmed by Bioanalyzer before conducting the assay. Around 20 amol of synthetic spike-in RNA oligo (ath-miR-159a, see Supplementary file 3) was added to each EV sample prior to RNA extraction and used as a normalization factor in downstream EV miRNA data analysis. Raw read data from the nCounter microRNA assay were analyzed as described in the nCounter Expression Data Analysis Guide (NanoString Technologies). Data normalization for cellular miRs was perfomed using the geometric mean of the top 100 expressed miRNAs. Only miRs that passed signal-to-noise cut-off values and that were consistently detected in either cellular or EV fractions (274 and 74 miRNAs, respectively) throughout all samples, were included in the analysis and plotted using R software. Normalized miRNA counts (Supplementary file 7) from SMPD3 KO or ARHGEF18 KO samples were divided by control miRNA counts from independent experiments (n = 2) and plotted as percent of control miRNA abundance.

Generation of CRISPR-KO cell lines for validation experiments--Polyclonal, CRISPR-KO cell lines were generated by transducing K562 stably expressing SFFV-Cas9-BFP with Lentiviral vectors encoding hit-targeting sgRNAs; at least two different sgRNAs per hit were tested. Cultures were incubated for at least 7–10 d with the sgRNAs to ensure adequate depletion of target-protein levels before conducting experiments. TIDE analysis (https://tide.nki.nl/) was performed to assess CRISPR deletions. Briefly, genomic DNA was extracted from cells using a QIAamp DNA mini kit (Qiagen). A 500–700 bp region containing the sgRNA cut site was amplified by PCR using GoTaq Polymerase (Promega). PCR amplification products were separated on a 0.75% TAE-agarose gel, from which amplicon bands were excised, and DNA was purified from the gel slice using a QIAquick Gel Extraction kit (Qiagen). Sanger sequencing of PCR products followed by trace decomposition analysis with TIDE software (https://tide.nki.nl/) was used to determine indel frequency in CRISPR-KO cell lines compared with control cells expressing a control sgRNA (targeting a genomic safe-harbor region). For validation experiments, sgRNA-expressing cell lines that showed higher degree of knockout efficiency as determined by either TIDE and/or immunoblot analysis were used (Figure 5—figure supplement 1).

Wnt signaling--For Wnt3a stimulation, K562 cells were starved overnight in serum-free conditions. The next day, the medium was replaced with fresh EV-free RPMI supplemented with 100 ng/ml recombinant Wnt3a protein (R and D Systems), or Wnt3a combined with 200 ng/ml recombinant DKK1 protein (R and D Systems) and EVs were collected 24 hr later. The GSK3 specific inhibitor, CHIR99021 (Selleckchem) and LiCl were used at 10 µM and 10 mM, respectively for 24 hr before collection of EVs from the medium. Quantitation of immunoblots from each of these conditions was normalized to account for cell viability, as measured by flow cytometry (BD Accuri). C59 Porcupine inhibitor was the generous gift of Dr. Roel Nusse, Stanford University.

Functional Network analysis--Top hits were collectively queried in STRING (https://string-db.org/) to search for gene interactions according to co-expression, experimental or curated database sources. The resulting STRING file reporting pair-wise gene interactions was imported into Cytoscape software (Shannon et al., 2003) to create a visually comprehensive functional interactome. Interactions were represented as edges whose thickness was proportional to a calculated combined score derived from STRING analysis. The final Cytoscape map was further manually curated to include additional screen hits (nodes) that despite not being reported to interact, could still be clustered in a common functional category revealed in the screen.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Albert Lu

   Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   alulopez@stanford.edu

   Competing interests

   No competing interests declared

2. Paulina Wawro

   Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

   Contribution

   Investigation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

3. David W Morgens

   Department of Genetics, Stanford University School of Medicine, Stanford, United States

   Contribution

   Data curation, Software, Formal analysis, Validation, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

4. Fernando Portela

   Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

   Contribution

   Data curation, Software, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0238-9251

5. Michael C Bassik

   Department of Genetics, Stanford University School of Medicine, Stanford, United States

   Contribution

   Conceptualization, Resources, Data curation, Software, Funding acquisition, Methodology, Writing—review and editing

   Competing interests

   No competing interests declared

6. Suzanne R Pfeffer

   Department of Biochemistry, Stanford University School of Medicine, Stanford, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   pfeffer@stanford.edu

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0002-6462-984X

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